BACKGROUND
FasL is a 40 kD TNF family member membrane protein that induces apoptosis by binding to Fas, its cell-surface receptor. FasL is expressed on activated T and NK cells. Both Fas and FasL are thought to play an important role in the apoptotic processes that take place during T cell development. PharMingen offers three monoclonal antibodies specific for human Fas Ligand.
NOK-1 recognizes human FasL.1 It recognizes both the membrane bound (FasL) and soluble (sFasL) forms. L5178Y mouse T lymphoma cells expressing recombinant human FasL were used as immunogen. FasL and sFasL migrate at reduced molecular weights of 40 and 26 kD, respectively. However, the molecular weights observed in a particular sample may vary according to FasL and sFasL glycosylation and breakdown patterns as described in Tanaka et al.2 Applications include immunoprecipitation (1-2 µg/one million cells), flow cytometry,1 and ELISA.1 The Biotin conjugated format of NOK-1 is also available for flow cytometry (Cat. #65322X). NOK-1 has also been shown to neutralize the cytotoxic activity of FasL.1 The NA/LE format of NOK-1 (Cat. #65320C) should be used for all functional assays. Neutralization of FasL activity inhibits Fas-mediated killing. NOK-1 and a related human FasL clone, NOK-2 may give different profiles in neutralization assays.1 It is thought that NOK-1 and NOK-2 likely recognize different FasL epitopes. Neither NOK-1 nor NOK-2 are suggested for western blot analysis. Clone G247-4 (cat. #65431A) is recommended for western blot.
NOK-2 recognizes human FasL.1 It recognizes both the membrane bound (FasL) and soluble (sFasL) forms. L5178Y mouse T lymphoma cells expressing recombinant human FasL were used as immunogen. FasL and sFasL migrate at reduced molecular weights of 40 and 26 kD, respectively. However, the molecular weights observed in a particular sample may vary according to FasL and sFasL glycosylation and breakdown patterns as described in Tanaka et al.2 Applications include immunoprecipitation (1-2 µg/one million cells), and ELISA.1 NOK-2 has also been shown to neutralize the cytotoxic activity of FasL.1 The NA/LE format of NOK-2 (Cat. #65330C) should be used for all functional assays. Neutralization of FasL activity inhibits Fas-mediated killing. NOK-2 and a related human FasL clone, NOK-1 may give different profiles in neutralization assays.1 It is thought that NOK-1 and NOK-2 likely recognize different FasL epitopes. NOK-2 is not recommended for flow cytometry; rather we suggest using the Purified or Biotin conjugated format of NOK-1flow cytometric analysis. Neither NOK-1 nor NOK-2 are suggested for western blot analysis. Clone G247-4 (cat. #65431A) is recommended for western blot.
G247-4 recognizes human FasL. It recognizes both the membrane bound (FasL) and soluble (sFasL) forms. A recombinant protein containing the external domain of human FasL was used as immunogen. FasL and sFasL migrate at reduced molecular weights of 40 and 26 kD, respectively. However, the molecular weights observed in a particular sample may vary according to FasL and sFasL glycosylation and breakdown patterns as described in Tanaka et al.2 For example, FasL may migrate as a doublet of 40 and 42 kD. Applications include immunoprecipitation (1-2 µg/one million cells), western blot analysis (1-2 µg/ml), flow cytometric analysis (titrate between 0.06 and 1 µg/one million cells) and ELISA assays. In ELISA sandwich assays, we have used G247-4 as a capture antibody with biotinylated NOK-1 as the detecting antibody. This pair specifically detected native soluble FasL in supernatants from FasL transfected L5187Y cells. The supernatant from the untransfected parental line (negative control) did not give a signal.
| Cat. No. | Description | Clone | Size |
| 65320C | Purified anti-human Fas Ligand, NA/LE | NOK-1 | 0.25mg |
| 65321A | Purified anti-human Fas Ligand NOK-1 | 0.1mg | |
| 65322X | Biotinylated anti-human Fas Ligand | NOK-1 | 100 tests |
| 65330C | Purified anti-human Fas Ligand, NA/LE | NOK-2 | 0.25mg |
| 65331A | Purified anti-human Fas Ligand | NOK-2 | 0.1mg |
| 65431A | Purified anti-human Fas ligand | G247-4 | 0.1mg |
REFERENCES
1. Kayagaki, N., A. Kawasaki, T. Ebata, H. Ohmoto, S. Ikeda, S. Inoue, K. Yoshino, K. Okumura, and H. Yagita. 1995. Metalloproteinase mediated release of human Fas ligand. J. Exp. Med. 182:1777-1783.
2. Tanaka, M., T. Suda, T. Takahashi, and S. Nagata. 1995. Expression of the functional soluble form of human Fas ligand in activated lymphocytes. EMBO 14:1129-1135.
PharMingen also supplies a number of antibodies for Apoptosis research including:
* Please inquire for availability
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